Project summary The precise organization of axonal projections into topographic maps is crucial for brain function, espe- cially in sensory systems. An important developmental mechanism contributing to topographic map formation is pre-target axon sorting, whereby axons are pre-ordered en route to their target according to their identity and/or positional origin. In the visual system, for instance, axon sorting occurs along the optic tract, where dor- sal and ventral retinal axons segregate respectively into the ventral and dorsal branches of the tract before reaching their brain target. While pre-target axon sorting has an instructive role in topographic mapping, how it is established during development remains poorly understood. Using the unique transparency and accessibility of the zebrafish embryo, our studies have shown that topographic order along the optic tract is not established during initial axon guidance but instead achieved through the selective degeneration of missorted dorsal axons that have erroneously misrouted along the dorsal branch. Heparan Sulfate (HS), a glycosaminoglycan carried by Heparan Sulfate Proteoglycans (HSPGs), is required non-cell autonomously for this selective degeneration. Yet, several questions remain unsolved. How does HS instruct missorted dorsal axons to degenerate while preserving those correctly elongating along the ventral branch of the tract? Is there a specific HSPG involved? Which molecular pathway(s) does HS regulate? A key difference between missorted and correctly targeted dorsal axons is their proximity to ventral pioneer axons. While erroneously navigating along the dorsal branch, missorted dorsal axons appear in close contact with ventral axons that have already elongated. Moreover, our preliminary data show that the HSPG glypican-3 (Gpc3) is selectively expressed in ventral RGCs in the mature retina. Thus, we hypothesize that Gpc3-mediated trans-axonal signaling between ventral pioneer and missorted dorsal follower retinal axons triggers the degeneration of missorted dorsal axons to estab- lish pre-target topographic sorting in the visual system. We will test that hypothesis by characterizing the expression and localization of Gpc3 in ventral retinal ganglion cells and corresponding axons (Aim 1), and by testing the function of Gpc3 in optic tract sorting in a cell specific manner (Aim 2). Gpc3 at the surface of pio- neer axons may act as a guidance cue acting directly on missorted dorsal axons to trigger their degeneration, or as a modulating factor controlling sorting indirectly by regulating a trans-axonal signaling pathway. To gain insight into which signaling factors might be regulated by Gpc3, we will test whether the semaphorin-neuropilin- plexin pathway, which is known to regulate axon ordering in other systems, also contributes to optic tract sort- ing (Aim 3). Altogether, these studies will be the first to determine how trans-axonal signaling between pioneer and follower axons establishes pre-target topographic sorting in the visual system, thus addressing a major gap in our understanding of the molecular mechanisms controlling topographic map formation.